Unknown,Transcriptomics,Genomics,Proteomics

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Growth Hormone Receptor and IGF-I Deficiency is Associated with Resistance to DNA Damage and Very Low Cancer and Diabetes Incidence in Humans

ABSTRACT: Growth Hormone Receptor Deficiency (GHRD) in mice causes life span extension and a major increase in the portion of animals that die without detectable pathologies including cancer and insulin resistance. During a 22-year monitoring of a cohort of 0-88 year old Ecuadorian GHRD subjects with severe IGF-I deficiency developed we observed a single, non-lethal malignancy and no diabetes. To understand the mechanisms responsible for this low disease incidence we incubated Human Mammary Epithelial Cells (HMECs) with serum from either GHRD or control subjects. GHRD serum not only protected mammary cells against hydrogen peroxide-dependent DNA damage but also promoted cell death in severely damaged cells by a mechanism blocked by IGF-I. The gene expression or DNA damage profile in epithelial cells exposed to GHRD serum, fibroblasts lacking the IGF-I receptor, and long-lived yeast lacking homologs of IGF-I signaling genes point to stress resistance transcription factors and SOD2 as mediators of GHRD-associated protection. These results provide evidence for a role of GH and IGF-I deficiency in promoting healthy aging in humans. Primary Human Mammalian Epithelial Cells (HMECs), were cultured in HMEC medium (ScienCell) at 37oC and 5% CO2 in Poly-L-Lysine coated culture dishes (Sigma). Treatment consisted of cells being stimulated with HMEC basal medium containing either 15% GHRD serum or 15% control serum for 6 hours. Cells were then harvested and processed for RNA extraction.

ORGANISM(S): Homo sapiens

SUBMITTER: Kevin Becker

PROVIDER: E-GEOD-21980 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Molecular distinctions between the stasis and telomere attrition senescence barriers in cultured human mammary epithelial cells Normal human epithelial cells in culture have generally shown a limited proliferative potential of ~10-40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in media composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50-60 population doublings, followed by p16(+), senescence-associated b-galactosidase(+) stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, i.e., cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo, in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean TRF length, and genomic stability, differed significantly between HMEC populations at the stasis vs. telomere attrition senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere attrition, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is Rb-mediated and independent of telomere length, while a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multi-lineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC. 48 samples from Human Mammary Epithelial cells which includes samples from four different individuals at different passage levels which includes prestasis,intermediate,post selection and agonesence stages of cell cycle.

Project description:Dysregulation of the epigenome is a common event in malignancy. However, deciphering the earliest cancer associated epigenetic events remains a challenge. Cancer epigenome studies to date have primarily utilised cancer cell lines or clinical samples, where it is difficult to identify the initial epigenetic lesions from those that occur over time. Here, we analysed the epigenome of normal Human Mammary Epithelial Cells (HMEC) and a matched variant cell population (vHMEC) that has escaped senescence and undergone partial carcinogenic transformation. Using this model system we sought to identify the earliest epigenetic changes that potentially occur during carcinogenesis. First we show that the transcriptome of vHMEC resembles that of basal-like breast cancer. Moreover, in vHMEC there is significant deregulation of MYC, p53, EZH2/polycomb, the Aryl Hydrocarbon Receptor (AHR) and miRNAs-143, 145, 199a and 519a at the transcriptional level. Second, we find that vHMEC exhibit genome-wide changes in DNA methylation affecting key cancer-associated pathways. Hypermethylation predominately impacted gene promoters (particularly those targeted by AHR and TP53) and polycomb associated loci, whereas hypomethylation frequently affected enhancers. Next we show that long range epigenetic deregulation occurred in vHMEC involving concordant change in chromatin modification and gene expression across ~0.5-1Mb regions. Finally, we demonstrate that the DNA methylation changes we observe in vHMECs, occur in basal-like breast cancer (notably FOXA1 hypermethylation).. Overall our results suggest that the first steps of carcinogenesis are associated with a co-ordinated deregulation of DNA methylation and chromatin modification spanning a range of genomic loci potentially targeted by key transcription factors and a corresponding deregulation of transcriptional networks. H3K27me3 ChIP-chip was performed on HMEC and vHMEC from 4 donors, H3K9ac was performed on cells from 2 donors. Both H3K27me3 and H3K9ac ChIP was performed on two time points for vHMEC and used the same input materia.

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Growth Hormone Receptor and IGF-I Deficiency is Associated with Resistance to DNA Damage and Very Low Cancer and Diabetes Incidence in Humans

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